Gastric cancer biomarker ENO3 and detection reagent thereof

Abstract

The application discloses a gastric cancer biomarker ENO3 and a detection reagent thereof, belongs to the technical field of biological medicine, and particularly relates to a detection reagent of a gastric cancer marker; the detection reagent comprises an upstream primer and a downstream primer of the gastric cancer marker and modified immunomagnetic beads; the gastric cancer marker is enolase 3. The application finds and verifies that the gastric cancer marker enolase 3 promotes glycolysis of gastric cancer cells, activates a WNT / beta-catenin signal pathway and up-regulates expression of matrix metalloproteinase, and promotes malignant transformation of gastric cancer; meanwhile, the structure of the immunomagnetic beads targeting the marker is optimized, the capture efficiency of the immunomagnetic beads on trace tumor cells in peripheral blood is improved, the signal strength of subsequent marker detection is enhanced, and an important tool for improving the positive detection rate of gastric cancer detection based on peripheral blood is provided.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a gastric cancer biomarker ENO3 and its detection reagent. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors worldwide and a leading cause of cancer-related deaths. Despite significant advancements in radical gastrectomy, adjuvant chemoradiotherapy, and combined immunotherapy, the overall prognosis for gastric cancer patients remains poor due to persistent postoperative recurrence and distant metastasis. Therefore, in-depth research into the key genes and downstream molecular mechanisms of gastric cancer development and progression, and the design of effective targeted intervention strategies based on this research, is of great significance for improving patient survival and prognosis.

[0003] Abnormal energy metabolism is one of the fundamental characteristics of tumor cells. Even under oxygen-sufficient conditions, tumor cells tend to preferentially utilize aerobic glycolysis (i.e., the Warburg effect), producing large amounts of lactic acid. This metabolic pathway not only provides tumor cells with a rapid energy supply but also provides raw materials for their biosynthesis, thereby promoting malignant biological behaviors such as tumor cell proliferation, migration, and invasion. Enolase (ENO) is one of the key enzymes in the glycolytic pathway, responsible for catalyzing the interconversion between 2-phosphoglycerate and phosphoenolpyruvate. In mammals, there are three families of enolases: ENO1, ENO2, and ENO3. Studies have shown that ENO1 and ENO2 play a pro-cancer role in various tumors. However, research reports on the role of ENO3 in tumors are relatively few, and its role is tissue-specific. For example, ENO3 is considered a potential tumor suppressor gene in liver cancer; while in colorectal cancer, ENO3 is highly expressed and associated with poor patient prognosis. Currently, the role of ENO3 in gastric cancer is unclear, and systematic research is lacking.

[0004] Current technological solutions still face the following limitations and challenges: First, there is a lack of reliable early diagnostic biomarkers. Atypical early symptoms of gastric cancer, concealed pathological features, and limitations in imaging examinations all affect the accuracy of early diagnosis. Second, the specific role of ENO3 in gastric cancer remains unclear. Although ENO3 has been studied in other tumor types, its expression pattern, clinical significance, biological function, and mechanism of action in gastric cancer are still unknown, limiting its potential as a therapeutic target. Third, glycolysis-targeted therapy still faces significant obstacles. Currently used glycolysis inhibitors, such as 2-deoxyglucose (2-DG), have significant side effects and limited efficacy, making the development of highly effective and low-toxicity metabolic intervention drugs urgent. Therefore, constructing a kit that can accurately and sensitively detect ENO3 expression will not only help reveal its role in the development and progression of gastric cancer but also provide an important tool for early diagnosis and the formulation of targeted therapy strategies. Summary of the Invention

[0005] The purpose of this invention is to provide a gastric cancer biomarker ENO3 and its detection reagent. It not only provides a biomarker related to gastric cancer, but also improves the capture efficiency of trace tumor cells in peripheral blood by optimizing the structure of immunomagnetic beads targeting the gastric cancer biomarker ENO3, thereby enhancing the signal intensity of subsequent biomarker detection and providing an important tool for improving the positive detection rate of gastric cancer based on peripheral blood.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: A detection reagent for a gastric cancer marker includes: an upstream primer and a downstream primer for the gastric cancer marker; the gastric cancer marker is enolase 3; the upstream primer sequence is 5'-GAACTCCGAGATGGAGACAAAG-3', and its nucleotide sequence is shown in SEQ ID NO.1; the downstream primer sequence is 5'-GGACCTAGAGTCTTGTTGATGTG-3', and its nucleotide sequence is shown in SEQ ID NO.2.

[0007] This invention discovers and verifies that the gastric cancer marker enolase 3 (ENO3) promotes glycolysis in gastric cancer cells, while simultaneously activating the WNT / β-catenin signaling pathway and upregulating the expression of matrix metalloproteinases, thereby driving the malignant transformation of gastric cancer. ENO3 is not only highly expressed in gastric cancer tissues, but its expression level is also closely related to patient prognosis, laying an important foundation for the development of reagents or kits for early detection and prognostic assessment of gastric cancer.

[0008] Preferably, the detection reagent also includes modified immunomagnetic beads.

[0009] More preferably, the modified immunomagnetic beads are composite magnetic nanoparticles with oleic acid-modified Fe3O4 composite nanoparticles as the core and a polymer of methacrylic acid and ethyl 2-[[(butylamino)carbonyl]oxo]acrylate as the structural unit as the coating layer. The surface of the coating layer is chemically coupled with immobilized enolase 3 antibody.

[0010] More preferably, the preparation method of modified immunomagnetic beads includes first mixing alkenyl derivatives, divinylbenzene and sodium dodecyl sulfate, adding deionized water and oleic acid-modified Fe3O4 composite nanoparticles for ultrasonic dispersion, and then reacting with acetic acid and H2O2 solution under a nitrogen atmosphere to obtain modified magnetic beads; subsequently, using EDC-NHS coupling technology, enolase 3 antibody is coupled to the surface of the modified magnetic beads to obtain modified immunomagnetic beads; the alkenyl derivatives include methacrylic acid and ethyl 2-[[(butylamino)carbonyl]oxo]acrylate.

[0011] More preferably, the mass ratio of methacrylic acid and ethyl 2-[[(butylamino)carbonyl]oxo]acrylate is 1:0.5-2.

[0012] More preferably, the ratio of alkenyl derivative to divinylbenzene is 1g:0.01-0.1mL.

[0013] More preferably, the ratio of divinylbenzene to sodium dodecyl sulfate is 1 mL: 0.2-0.5 g.

[0014] More preferably, the mass ratio of sodium dodecyl sulfate and oleic acid-modified Fe3O4 composite nanoparticles is 1:0.5-2.

[0015] More preferably, the volume ratio of divinylbenzene to acetic acid is 1:0.1-0.5.

[0016] More preferably, the mass concentration of the H2O2 solution is 20-40%, and the volume ratio of divinylbenzene to H2O2 solution is 1:0.1-0.5.

[0017] Preferably, the detection reagent for the gastric cancer biomarker ENO3 includes 5-15 mL of EDTA anticoagulant, 100-500 μL of modified immunomagnetic beads, 10-50 mL of washing buffer, 100-500 μL of buffer solution, 0.5-2 mL of cell dissociation solution, 1-5 parts by weight of total RNA extraction kit, 1-5 parts by weight of SYBR Green one-step qRT-PCR kit, 1-2 OD of upstream primer and 1-2 OD of downstream primer for the gastric cancer biomarker ENO3.

[0018] More preferably, the washing solution is a phosphate buffer solution containing 0.01-0.2% bovine serum albumin.

[0019] More preferably, the buffer solution is a phosphate buffer solution containing 0.01-0.1% bovine serum albumin.

[0020] More preferably, the cell dissociation solution is an Accutase enzyme solution.

[0021] More preferably, the upstream primer sequence of the gastric cancer biomarker ENO3 is 5'-GAACTCCGAGATGGAGACAAAG-3', and its nucleotide sequence is shown in SEQ ID NO.1.

[0022] More preferably, the downstream primer sequence is 5'-GGACCTAGAGTCTTGTTGATGTG-3', and its nucleotide sequence is shown in SEQ ID NO.2.

[0023] More preferably, the preparation method of modified immunomagnetic beads is as follows: S1. Mix alkenyl derivatives, divinylbenzene and sodium dodecyl sulfate, add deionized water and oleic acid modified Fe3O4 composite nanoparticles and ultrasonically disperse for 20-60 min. Add acetic acid and H2O2 solution under nitrogen atmosphere and react at 40-60℃ for 8-16 h. After the reaction is completed, cool to room temperature and centrifuge at 5000-10000 rpm / min for 5-15 min. Discard the supernatant and wash with deionized water 1-5 times to obtain modified magnetic beads.

[0024] S2. The modified magnetic beads were ultrasonically dispersed in phosphate buffer, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added for activation for 20-40 min. After magnetic separation, the supernatant was discarded, ENO3 antibody solution was added and reacted at 0-10℃ for 8-16 h. Ethanolamine solution was added to block the reaction for 1-3 h. The beads were washed 1-5 times with phosphate buffer, and after magnetic separation, buffer was added to obtain the modified immunomagnetic beads.

[0025] More preferably, in step S1, the alkenyl derivative is at least two of the following: methacrylic acid, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate, N-allyl-P-methoxyaniline, and methyl 3-allyl-2-hydroxybenzoate.

[0026] More preferably, in step S1, the alkenyl derivative is methacrylic acid and ethyl 2-[[(butylamino)carbonyl]oxo]acrylate; the mass ratio of methacrylic acid to ethyl 2-[[(butylamino)carbonyl]oxo]acrylate is 1:0.5-2. This invention modifies the surface of Fe3O4 particles with a polymer consisting of methacrylic acid and ethyl 2-[[(butylamino)carbonyl]oxo]acrylate as structural units, and forms modified immunomagnetic beads by conjugating antibodies. This may introduce more active groups, providing abundant reaction sites for antibody conjugation. Upon contact with tumor cells, multiple antibodies can bind to antigen molecules, thereby enhancing the capture ability of tumor cells and effectively improving the positive detection rate in the detection system.

[0027] More preferably, in step S1, the alkenyl derivative is methacrylic acid, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate, and N-allyl-P-methoxyaniline; the mass ratio of methacrylic acid to ethyl 2-[[(butylamino)carbonyl]oxo]acrylate is 1:0.5-2; and the mass ratio of methacrylic acid to N-allyl-P-methoxyaniline is 1:0.1-0.25. This invention, by using ethyl 2-[[(butylamino)carbonyl]oxo]acrylate and N-allyl-P-methoxyaniline in combination, may help enhance the stability of the polymer chain binding to the antibody and maintain the activity of the antibody binding to the antigen through intermolecular interactions, thereby improving the capture ability of tumor cells, reducing background interference, and effectively increasing the positive detection rate in the detection system.

[0028] More preferably, in step S1, the alkenyl derivative is methacrylic acid, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate, N-allyl-P-methoxyaniline, and methyl 3-allyl-2-hydroxybenzoate; the mass ratio of methacrylic acid to ethyl 2-[[(butylamino)carbonyl]oxo]acrylate is 1:0.5-2; the mass ratio of methacrylic acid to N-allyl-P-methoxyaniline is 1:0.1-0.25; and the mass ratio of methacrylic acid to methyl 3-allyl-2-hydroxybenzoate is 1:0.3-1. The present invention further uses methyl 3-allyl-2-hydroxybenzoate, which may further provide abundant active sites, achieving high-density antibody immobilization, helping to further enhance the recognition and capture of tumor cells, and thus further improving the positive detection rate in the biomarker detection system.

[0029] More preferably, in step S1, the ratio of alkenyl derivative to divinylbenzene is 1g:0.01-0.1mL.

[0030] More preferably, in step S1, the ratio of divinylbenzene to sodium dodecyl sulfate is 1 mL: 0.2-0.5 g.

[0031] More preferably, in step S1, the ratio of sodium dodecyl sulfate to deionized water is 1g:50-200mL.

[0032] More preferably, in step S1, the mass ratio of sodium dodecyl sulfate and oleic acid-modified Fe3O4 composite nanoparticles is 1:0.5-2.

[0033] More preferably, the volume ratio of divinylbenzene to acetic acid in step S1 is 1:0.1-0.5.

[0034] More preferably, the mass concentration of the H2O2 solution in step S1 is 20-40%.

[0035] More preferably, the volume ratio of divinylbenzene to H2O2 solution in step S1 is 1:0.1-0.5.

[0036] More preferably, in step S2, the ratio of modified magnetic beads to phosphate buffer is 1g:20-100mL.

[0037] More preferably, in step S2, the mass ratio of the modified magnetic beads to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:0.5-1.

[0038] More preferably, in step S2, the mass ratio of modified magnetic beads to N-hydroxysuccinimide is 1:0.2-1.

[0039] More preferably, the ENO3 antibody solution in step S2 is a phosphate buffer containing 0.5-2 mg / mL of ENO3 antibody.

[0040] More preferably, in step S2, the ratio of modified magnetic beads to ENO3 antibody solution is 1g:5-20mL.

[0041] More preferably, the concentration of the ethanolamine solution in step S2 is 0.05-0.2 mol / L.

[0042] More preferably, the volume ratio of ENO3 antibody solution to ethanolamine solution in step S2 is 1:0.5-2.

[0043] More preferably, the buffer in step S2 is a phosphate buffer containing 0.05-0.2% bovine serum albumin and 0.01-0.1% sodium azide.

[0044] More preferably, in step S2, the ratio of modified magnetic beads to buffer solution is 1g:20-100mL.

[0045] Preferably, the detection method for the gastric cancer marker ENO3 is as follows: S1. Blood sample collection: Peripheral blood samples are drawn and EDTA anticoagulant is added to obtain blood samples.

[0046] S2. Tumor cell enrichment: Mix the blood sample with modified immunomagnetic beads for 20-60 min, then place it on a magnetic rack for 2-10 min, discard the suspension, wash 1-5 times with washing buffer, remove from the magnetic rack, add buffer again and mix well, centrifuge at 300-1000 rpm for 2-10 min, retain the precipitate, add cell dissociation solution and incubate for 2-10 min, then place on a magnetic rack for 2-10 min, collect the supernatant, and collect the tumor cells.

[0047] S3. Detection of gastric cancer biomarkers: Total RNA was extracted from tumor cells using a total RNA extraction kit, and the expression of the gastric cancer biomarker ENO3 was detected using the SYBR Green one-step qRT-PCR kit. The total RNA extraction kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., and the SYBR Green one-step qRT-PCR kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The upstream primer sequence for qRT-PCR detection of the gastric cancer biomarker ENO3 was 5'-GAACTCCGAGATGGAGACAAAG-3', and its nucleotide sequence is shown in SEQ ID NO.1; the downstream primer sequence was 5'-GGACCTAGAGTCTTGTTGATGTG-3', and its nucleotide sequence is shown in SEQ ID NO.2. Using GAPDH as a reference gene, the upstream primer sequence for GAPDH was 5'-GCATCCACTGGTGCTGCC-3', and its nucleotide sequence is shown in SEQ ID NO.3; the downstream primer sequence was 5'-TCATCATACTTGGCAGGTTTC-3', and its nucleotide sequence is shown in SEQ ID NO.4. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0048] More preferably, the EDTA anticoagulant in step S1 is EDTA.2K anticoagulant (10×).

[0049] More preferably, in step S1, the volume ratio of peripheral blood sample to EDTA anticoagulant is 1:5-15.

[0050] More preferably, in step S2, the volume ratio of blood sample to modified immunomagnetic beads is 1:0.1-0.5.

[0051] More preferably, the washing solution in step S2 is a phosphate buffer solution containing 0.05-0.2% bovine serum albumin.

[0052] More preferably, the buffer solution in step S2 is a phosphate buffer solution containing 0.02-0.1% bovine serum albumin.

[0053] More preferably, the volume ratio of blood sample to buffer solution in step S2 is 1:0.1-0.5.

[0054] More preferably, the cell dissociation solution in step S2 is an Accutase enzyme solution.

[0055] More preferably, in step S2, the volume ratio of blood sample to cell dissociation solution is 1:0.5-2.

[0056] This invention utilizes modified immunomagnetic beads to enrich trace amounts of tumor cells in peripheral blood samples from gastric cancer patients, followed by qRT-PCR detection of the gastric cancer marker ENO3. Therefore, it offers the following advantages: The modified immunomagnetic beads prepared in this invention possess highly efficient and specific capture capabilities for tumor cells. This significantly improves the enrichment efficiency of trace tumor cells in peripheral blood samples, thereby effectively enhancing the positive detection rate of gastric cancer markers and providing technical support for the early detection of gastric cancer. Attached Figure Description

[0057] Figure 1 This shows the representation of ENO3 in the TCGA database.

[0058] Figure 2 This shows the representation of ENO3 in the ACRG database.

[0059] Figure 3 This shows the mRNA expression of ENO3 in cohort 1.

[0060] Figure 4 The protein expression level of ENO3 in cohort 2 is shown.

[0061] Figure 5 The results are from the KM-Plot database. The black line represents samples with low ENO3 expression, with a total of 218 samples; the red line represents samples with high ENO3 expression, with a total of 657 samples.

[0062] Figure 6 To stabilize the expression of ENO3 protein in MKN45 cell lines transfected with knockdown of ENO3.

[0063] Figure 7 To stabilize the expression of ENO3 protein in SGC-7901 cell lines with knocked-down ENO3.

[0064] Figure 8 To stabilize the proliferation of MKN45 cell lines transfected with knockdown of ENO3.

[0065] Figure 9 The results of plate cloning of SGC-7901 cell lines with stable ENO3 knockdown transfection.

[0066] Figure 10 Results of plate cloning of MKN45 cell lines with stable ENO3 knockdown transfection.

[0067] Figure 11 To stabilize the migration results of SGC-7901 cell lines transfected with knocked-down ENO3.

[0068] Figure 12 To stabilize the migration results of MKN45 cell lines transfected with knocked-down ENO3.

[0069] Figure 13 To stabilize the invasion results of SGC-7901 cell lines transfected with knocked-down ENO3.

[0070] Figure 14 To stabilize the invasion results of MKN45 cell lines transfected with knockdown ENO3.

[0071] Figure 15 The baseline expression level of ENO3 in gastric cancer cell lines.

[0072] Figure 16 The expression of ENO3 protein in the AGS cell line that overexpresses ENO3.

[0073] Figure 17 The expression of ENO3 protein in HGC cell lines that overexpress ENO3.

[0074] Figure 18 To assess the proliferation capacity of AGS cell lines overexpressing ENO3.

[0075] Figure 19 To assess the proliferation capacity of HGC cell lines overexpressing ENO3.

[0076] Figure 20 The migration results of AGS cell lines overexpressing ENO3.

[0077] Figure 21 The migration results of HGC cell lines overexpressing ENO3.

[0078] Figure 22 The results of invasion of AGS cell lines overexpressing ENO3.

[0079] Figure 23 The results of invasion of HGC cell lines overexpressing ENO3.

[0080] Figure 24 To stabilize lactate levels in cell lines transfected with knocked-down ENO3.

[0081] Figure 25 To stabilize the ATP content in cell lines transfected with knocked-down ENO3.

[0082] Figure 26 The lactate content in cell lines overexpressing ENO3.

[0083] Figure 27 The ATP content in cell lines overexpressing ENO3.

[0084] Figure 28 This indicates the expression status of target genes.

[0085] Figure 29 The expression and phosphorylation levels of β-catenin and GSK-3β proteins.

[0086] Figure 30 To stabilize the expression of MMP genes in the SGC-7901 cell line with ENO3 knockdown.

[0087] Figure 31 The expression of MMP genes in HGC cell lines that overexpress ENO3.

[0088] Figure 32 The expression of MMP genes in the SGC-7901 cell line that overexpresses ENO3.

[0089] Figure 33 To stabilize the expression of MMP2 protein in MKN45 cell lines transfected with knockdown of ENO3.

[0090] Figure 34 To stabilize the expression of MMP2 protein in SGC-7901 cell line with ENO3 knockdown after transfection.

[0091] Figure 35 The expression of MMP2 protein in the MKN45 cell line overexpressing ENO3.

[0092] Figure 36 The morphology of the modified immunomagnetic beads is shown. Detailed Implementation

[0093] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0094] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0095] Example 1: A biomarker for gastric cancer includes enolase 3 (ENO3).

[0096] Example 2: Methods for preparing modified immunomagnetic beads include, S1. Mix alkenyl derivatives, divinylbenzene and sodium dodecyl sulfate, add deionized water and oleic acid modified Fe3O4 composite nanoparticles and ultrasonically disperse for 30 min. Add acetic acid and H2O2 solution under nitrogen atmosphere and react at 50℃ for 12 h. After the reaction is completed, cool to room temperature, centrifuge at 8000 rpm / min for 10 min, discard the supernatant, and wash 3 times with deionized water to obtain modified magnetic beads. The alkenyl derivative is methyl methacrylate and ethyl 2-[[(butylamino)carbonyl]oxo]acrylate, wherein the mass ratio of methyl methacrylate to ethyl 2-[[(butylamino)carbonyl]oxo]acrylate is 1:1; the mass ratio of alkenyl derivative to divinylbenzene is 1g:0.02mL; the mass ratio of divinylbenzene to sodium dodecyl sulfate is 1mL:0.3g; the mass ratio of sodium dodecyl sulfate to deionized water is 1g:100mL; the mass ratio of sodium dodecyl sulfate to oleic acid-modified Fe3O4 composite nanoparticles is 1:1; the volume ratio of divinylbenzene to acetic acid is 1:0.25; the mass concentration of H2O2 solution is 30%, and the volume ratio of divinylbenzene to H2O2 solution is 1:0.25. The CAS number of ethyl 2-[[(butylamino)carbonyl]oxo]acrylate is 63225-53-6, and its chemical structure is shown in Formula I. .

[0097] S2. The modified magnetic beads were ultrasonically dispersed in phosphate buffer, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added for activation for 30 min. After magnetic separation, the supernatant was discarded, ENO3 antibody solution was added and reacted at 4℃ for 12 h. Ethanolamine solution was added to block the reaction for 2 h. The beads were washed 3 times with phosphate buffer, and after magnetic separation, buffer was added to obtain the modified immunomagnetic beads. The volume ratio of modified magnetic beads to phosphate buffer was 1 g: 50 mL; the mass ratio of modified magnetic beads to 1-ethyl-(3-dimethylaminopropyl)carbodiimide was 1:0.7; the mass ratio of modified magnetic beads to N-hydroxysuccinimide was 1:0.5; the ENO3 antibody solution was a phosphate buffer containing 1 mg / mL ENO3 antibody, and the volume ratio of modified magnetic beads to ENO3 antibody solution was 1 g: 10 mL; the concentration of ethanolamine solution was 0.1 mol / L, and the volume ratio of ENO3 antibody solution to ethanolamine solution was 1:1; the buffer solution was a phosphate buffer containing 0.1% bovine serum albumin and 0.05% sodium azide, and the volume ratio of modified magnetic beads to buffer solution was 1 g: 50 mL.

[0098] The detection reagent for the gastric cancer biomarker ENO3 includes: 9 mL of EDTA anticoagulant, 250 μL of modified immunomagnetic beads, 30 mL of washing buffer, 200 μL of buffer, 1 mL of cell dissociation solution, 1 part by weight of total RNA extraction kit, 1 part by weight of SYBR Green one-step qRT-PCR kit, 1 OD of upstream primer and 1 OD of downstream primer for the gastric cancer biomarker ENO3. The washing buffer was a phosphate-buffered saline solution containing 0.1% bovine serum albumin; the buffer solution was a phosphate-buffered saline solution containing 0.05% bovine serum albumin; the cell dissociation solution was Accutase enzyme solution, purchased from Thermo Fisher Scientific; the total RNA extraction kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.; the SYBR Green one-step qRT-PCR kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the upstream primer sequence for the gastric cancer biomarker ENO3 was 5'-GAACTCCGAGATGGAGACAAAG-3', and its nucleotide sequence is shown in SEQ ID NO.1; the downstream primer sequence was 5'-GGACCTAGAGTCTTGTTGATGTG-3', and its nucleotide sequence is shown in SEQ ID NO.2.

[0099] Methods for detecting the gastric cancer biomarker ENO3 include, S1. Blood Sample Collection: Peripheral blood samples were collected and EDTA anticoagulant was added to obtain blood samples. The EDTA anticoagulant was EDTA.2K anticoagulant (10×), purchased from Beijing Solarbio Science & Technology Co., Ltd. The volume ratio of peripheral blood sample to EDTA anticoagulant was 1:9. S2. Tumor Cell Enrichment: Blood samples were mixed with modified immunomagnetic beads for 30 min, then placed on a magnetic rack for 5 min. The suspension was discarded, and the samples were washed three times with washing buffer. The samples were removed from the rack, and buffer was added again for mixing. The mixture was centrifuged at 500 rpm for 5 min, and the precipitate was retained. Cell dissociation buffer was added and incubated for 5 min, then placed on a magnetic rack for 5 min. The supernatant was collected, yielding tumor cells. The volume ratio of blood sample to modified immunomagnetic beads was 1:0.25; the washing buffer was a phosphate-buffered saline solution containing 0.1% bovine serum albumin; the buffer solution was a phosphate-buffered saline solution containing 0.05% bovine serum albumin, with a blood sample to buffer volume ratio of 1:0.2; the cell dissociation buffer was Accutase enzyme solution, purchased from Thermo Fisher Scientific, with a blood sample to cell dissociation buffer volume ratio of 1:1.

[0100] S3. Detection of gastric cancer biomarkers: Total RNA was extracted from tumor cells using a total RNA extraction kit, and the expression of the gastric cancer biomarker ENO3 was detected using the SYBR Green one-step qRT-PCR kit. The total RNA extraction kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., and the SYBR Green one-step qRT-PCR kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The upstream primer sequence for qRT-PCR detection of the gastric cancer biomarker ENO3 was 5'-GAACTCCGAGATGGAGACAAAG-3', and its nucleotide sequence is shown in SEQ ID NO.1; the downstream primer sequence was 5'-GGACCTAGAGTCTTGTTGATGTG-3', and its nucleotide sequence is shown in SEQ ID NO.2. Using GAPDH as a reference gene, the upstream primer sequence for GAPDH was 5'-GCATCCACTGGTGCTGCC-3', and its nucleotide sequence is shown in SEQ ID NO.3; the downstream primer sequence was 5'-TCATCATACTTGGCAGGTTTC-3', and its nucleotide sequence is shown in SEQ ID NO.4. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0101] Example 3: The method for preparing modified immunomagnetic beads differs from Example 2 except that in step S1, the alkenyl derivative components are changed to: the alkenyl derivative is methacrylic acid, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate and N-allyl-P-methoxyaniline, wherein the mass ratio of methacrylic acid and ethyl 2-[[(butylamino)carbonyl]oxo]acrylate is 1:1, and the mass ratio of methacrylic acid and N-allyl-P-methoxyaniline is 1:0.255, and other conditions are the same as in Example 2.

[0102] Example 4: The method for preparing modified immunomagnetic beads differs from Example 2 except that in step S1, the alkenyl derivative components are changed to: the alkenyl derivative is methacrylic acid, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate and N-allyl-P-methoxyaniline, wherein the mass ratio of methacrylic acid and ethyl 2-[[(butylamino)carbonyl]oxo]acrylate is 1:1, and the mass ratio of methacrylic acid and N-allyl-P-methoxyaniline is 1:0.1, and other conditions are the same as in Example 2.

[0103] The detection reagent for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0104] The detection method for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0105] Example 5: The method for preparing modified immunomagnetic beads differs from Example 2 except that in step S1, the alkenyl derivative components are changed to: the alkenyl derivative is methacrylic acid, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate and N-allyl-P-methoxyaniline, wherein the mass ratio of methacrylic acid and ethyl 2-[[(butylamino)carbonyl]oxo]acrylate is 1:1, and the mass ratio of methacrylic acid and N-allyl-P-methoxyaniline is 1:0.05, and other conditions are the same as in Example 2.

[0106] The detection reagent for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0107] The detection method for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0108] Example 6: The method for preparing modified immunomagnetic beads differs from Example 2 except that in step S1, the alkenyl derivative components are changed to: the alkenyl derivative is methacrylic acid, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate and N-allyl-P-methoxyaniline, wherein the mass ratio of methacrylic acid and ethyl 2-[[(butylamino)carbonyl]oxo]acrylate is 1:1, and the mass ratio of methacrylic acid and N-allyl-P-methoxyaniline is 1:0.5, and other conditions are the same as in Example 2.

[0109] The detection reagent for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0110] The detection method for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0111] Example 7: The method for preparing modified immunomagnetic beads differs from Example 2 except that in step S1, the alkenyl derivative components are changed to: ethyl methacrylate, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate, N-allyl-P-methoxyaniline, and methyl 3-allyl-2-hydroxybenzoate, wherein the mass ratio of methacrylate to ethyl 2-[[(butylamino)carbonyl]oxo]acrylate is 1:1, the mass ratio of methacrylate to N-allyl-P-methoxyaniline is 1:0.25, and the mass ratio of methacrylate to methyl 3-allyl-2-hydroxybenzoate is 1:0.6; other conditions are the same as in Example 2.

[0112] The detection reagent for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0113] The detection method for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0114] Comparative Example 1: The detection reagent for the gastric cancer biomarker ENO3 includes 9 mL of EDTA anticoagulant, 1 part by weight of a total RNA extraction kit, 1 part by weight of a SYBR Green one-step qRT-PCR kit, 1 OD of an upstream primer and 1 OD of a downstream primer for the gastric cancer biomarker ENO3. The total RNA extraction kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.; the SYBR Green one-step qRT-PCR kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the upstream primer sequence for the gastric cancer biomarker ENO3 is 5'-GAACTCCGAGATGGAGACAAAG-3', and its nucleotide sequence is shown in SEQ ID NO.1; the downstream primer sequence is 5'-GGACCTAGAGTCTTGTTGATGTG-3', and its nucleotide sequence is shown in SEQ ID NO.2.

[0115] The detection method for the gastric cancer biomarker ENO3 is the same as that in Example 2, except that step S2 is not performed.

[0116] Comparative Example 2: The preparation method of modified immunomagnetic beads is the same as that in Example 2, except that step S1 is not performed and the modified magnetic beads in step S2 are replaced with carboxyl magnetic beads purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0117] The detection reagent for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0118] The detection method for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0119] Comparative Example 3: The preparation method of the modified immunomagnetic beads is the same as that in Example 2, except that step S1 is not performed and the modified magnetic beads in step S2 are replaced with amino magnetic beads. The amino magnetic beads were purchased from Shanghai Beyotime Biotechnology Co., Ltd. All other conditions are the same as in Example 2.

[0120] The detection reagent for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0121] The detection method for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0122] Comparative Example 4: The preparation method of the modified immunomagnetic beads is the same as that in Example 2, except that in step S1, the alkenyl derivative component is changed to methacrylic acid, and the other conditions are the same as in Example 2.

[0123] The detection reagent for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0124] The detection method for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0125] Comparative Example 5: The method for preparing modified immunomagnetic beads differs from Example 2 except that in step S1, the alkenyl derivative is changed to consist of methacrylic acid and N-allyl-P-methoxyaniline, with a mass ratio of methacrylic acid to N-allyl-P-methoxyaniline of 1:0.25. All other conditions are the same as in Example 2.

[0126] The detection reagent for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0127] The detection method for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0128] Comparative Example 6: The method for preparing modified immunomagnetic beads differs from Example 2 except that in step S1, the alkenyl derivative is changed to consist of methacrylic acid and methyl 3-allyl-2-hydroxybenzoate, with a mass ratio of methacrylic acid to methyl 3-allyl-2-hydroxybenzoate of 1:0.6. All other conditions are the same as in Example 2.

[0129] The detection reagent for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0130] The detection method for the gastric cancer biomarker ENO3 is the same as in Example 2, except that the modified immunomagnetic beads are replaced with the modified immunomagnetic beads prepared in this example.

[0131] Experimental Example 1: Paired and unpaired analyses were performed using gastric cancer data from the Cancer Genome Atlas (TCGA) and the Asian Cancer Research Group (ACRG) databases to determine the mRNA expression levels of the ENO3 gene in gastric cancer tissues. The results are as follows: Figure 1 and Figure 2 As shown, in both the TCGA and ACRG databases, paired and unpaired analyses revealed that the mRNA expression level of ENO3 in gastric cancer tissues was higher than that in normal gastric tissues.

[0132] Validation was performed using samples from two gastric cancer cohorts at Zhejiang Cancer Hospital. Cohort 1 contained 54 pairs of gastric cancer tissue and paired normal tissue samples, and Cohort 2 contained 43 pairs of gastric cancer tissue and paired normal tissue samples. The mRNA expression level of the ENO3 gene in Cohort 1 and the protein expression level of ENO3 in Cohort 2 were obtained. The results are as follows: Figure 3 As shown, in the 54 pairs of samples in cohort 1, the mRNA expression level of ENO3 was increased in gastric cancer tissues compared with paired normal tissues. The results are as follows... Figure 4 As shown, in the 43 pairs of samples in cohort 2, the protein expression level of ENO3 in gastric cancer tissues was significantly higher than that in the paired normal tissues.

[0133] The relationship between ENO3 expression and overall patient survival was analyzed using the KM-Plot database, which contains 218 samples with low ENO3 expression and 657 samples with high ENO3 expression. Results are as follows: Figure 5As shown, high ENO3 expression was significantly associated with poorer overall survival in patients, with a hazard ratio (HR) of 1.62 and a P value of <0.001.

[0134] This indicates that ENO3 is highly expressed in gastric cancer tissues and suggests a poor prognosis.

[0135] Experimental Example 2: Using lentiviral shRNA technology, stable ENO3 knockdown cell lines were constructed in gastric cancer cell lines. When using MKN45 cells, a stable ENO3 knockdown cell line was obtained; when using SGC-7901 cells, a stable ENO3 knockdown cell line was obtained. The stable ENO3 knockdown cell lines were divided into three groups: a blank control group (sh-con), experimental group 1 (sh1-ENO3), and experimental group 2 (sh2-ENO3). The blank control group (sh-con) consisted of cell lines without stable ENO3 knockdown transfection, while experimental groups 1 (sh1-ENO3) and 2 (sh2-ENO3) were two replicates of the stable ENO3 knockdown cell lines.

[0136] Western blot (WB) was used to verify the knockdown efficiency, with β-actin as the reference gene. The results are as follows: Figure 6 and Figure 7 It can be seen that stable cell lines with ENO3 knockdown were constructed in both MKN45 cells and SGC-7901 cells.

[0137] The blank control group (sh-con), experimental group 1 (sh1-ENO3), and experimental group 2 (sh2-ENO3) of MKN45 cell lines stably transfected with knockdown of ENO3 were cultured for 5 days. Samples were taken on days 1, 2, 3, 4, and 5 of culture, and cell proliferation capacity was measured using the CCK-8 assay.

[0138] The results are as follows Figure 8 As shown, in the early stage of culture, there was no significant difference in the fold increase of cell proliferation among the sh-con, sh1-ENO3, and sh2-ENO3 groups; however, from day 3 of culture, the fold increase of cell proliferation in the sh-con group was significantly higher than that in the sh1-ENO3 and sh2-ENO3 groups. This indicates that knocking down the ENO3 gene can significantly inhibit the proliferation of gastric cancer cells.

[0139] The plate cell colony formation assay was used to detect the single cell colony proliferation levels of the blank control group (sh-con), experimental group 1 (sh1-ENO3), and experimental group 2 (sh2-ENO3) of the SGC-7901 cell line stably transfected with knockdown ENO3. After the plate cell colony formation assay was completed, the cells were observed and photographed under a microscope to quantify the cell colony formation level of each group.

[0140] The results are as follows Figure 9 As shown, the clonogenic level in the sh-con group was higher than that in the sh1-ENO3 and sh2-ENO3 groups. This indicates that knocking down the ENO3 gene can significantly inhibit the proliferation of single-cell clones of gastric cancer cells.

[0141] The colony formation assay was used to detect the colony formation levels of the blank control group (sh-con), experimental group 1 (sh1-ENO3), and experimental group 2 (sh2-ENO3) of the MKN45 cell line stably transfected with knockdown ENO3. After the soft agar cell colony formation assay was completed, the cells were observed and photographed under a microscope to quantify the colony formation level of each group.

[0142] The results are as follows Figure 10 As shown, the clonogenic level in the sh-con group was higher than that in the sh1-ENO3 and sh2-ENO3 groups. This indicates that knocking down the ENO3 gene can significantly inhibit the clonogenic ability of gastric cancer cells.

[0143] The Transwell assay was used to detect the migration and invasion abilities of cell lines stably transfected with knocked-down ENO3 (sh-con group, sh1-ENO3 group and sh2-ENO3 group). After the Transwell assay was completed, the cells were observed and photographed under a microscope, and the migration and invasion of cells were quantitatively analyzed.

[0144] The results are as follows Figure 11 and Figure 12 As shown, in the SGC-7901 cell line and the MKN45 cell line stably transfected with ENO3 knockdown, the number of cells migrating in the sh-con group was significantly higher than that in the sh1-ENO3 group and the sh2-ENO3 group. This indicates that knockdown of the ENO3 gene can significantly inhibit the cell migration ability of gastric cancer cells.

[0145] The results are as follows Figure 13 and Figure 14 As shown, in the SGC-7901 cell line and the MKN45 cell line stably transfected with ENO3 knockdown, the invasive cells in the sh-con group were significantly higher than those in the sh1-ENO3 group and the sh2-ENO3 group. This indicates that knockdown of the ENO3 gene can significantly inhibit the invasive ability of gastric cancer cells.

[0146] This demonstrates that knocking down the ENO3 gene can inhibit the proliferation, migration, and invasion of gastric cancer cells.

[0147] Experimental Example 3: The background expression level of ENO3 in gastric cancer cell lines AGS, MKN45, SGC, BGC and HGC was detected by Western blot (WB), with β-actin as the reference gene.

[0148] The results are as follows Figure 15 As shown, the basal expression levels of ENO3 in AGS cells and HGC cells are relatively low. Therefore, AGS cells and HGC cells were selected to construct cell lines overexpressing ENO3.

[0149] Using lentivirus-mediated gene overexpression, ENO3-overexpressing cell lines were constructed in gastric cancer cell lines. When using AGS cells, an ENO3-overexpressing AGS cell line was obtained; when using HGC cells, an ENO3-overexpressing HGC cell line was obtained. The ENO3-overexpressing cell lines were divided into two groups: a blank control group (oe-con) and an overexpression group (oe-ENO3). The blank control group (oe-con) consisted of cell lines that did not undergo ENO3 overexpression, while the overexpression group (oe-ENO3) consisted of cell lines that overexpressed ENO3.

[0150] Overexpression efficiency was verified using Western blot (WB), with β-actin as the reference gene. Results are as follows: Figure 16 and Figure 17 It can be seen that cell lines overexpressing ENO3 were constructed in both AGS cells and HGC cells.

[0151] Cells from the oe-con group and oe-ENO3 group of the ENO3 overexpressing cell line were cultured for 5 days, and samples were taken on days 1, 2, 3, 4 and 5 of culture, respectively. Cell proliferation capacity was measured using the CCK-8 assay.

[0152] The results are as follows Figure 18 and Figure 19 As shown, in the early stages of AGS cell culture, there was no significant difference in the fold increase of cell proliferation between the oe-con group and the oe-ENO3 group; however, on day 5 of culture, the fold increase of cell proliferation in the oe-ENO3 group was significantly higher than that in the oe-con group. Similarly, in the early stages of HGC cell culture, there was no significant difference in the fold increase of cell proliferation between the oe-con group and the oe-ENO3 group; however, on day 3 of culture, the fold increase of cell proliferation in the oe-ENO3 group was significantly higher than that in the oe-con group. This indicates that overexpression of the ENO3 gene can significantly enhance the proliferative capacity of gastric cancer cells.

[0153] Transwell assays were used to detect the migration and invasion abilities of cell lines overexpressing ENO3 (oe-con group and oe-ENO3 group). After the Transwell assays were completed, the cells were observed and photographed under a microscope, and the migration and invasion of cells were quantitatively analyzed.

[0154] The results are as follows Figure 20 and Figure 21 As shown, in both the AGS and HGC cell lines overexpressing ENO3, the number of cells migrating in the oe-ENO3 group was significantly higher than that in the oe-con group. This indicates that overexpression of the ENO3 gene can significantly enhance the cell migration ability of gastric cancer cells.

[0155] The results are as follows Figure 22 and Figure 23 As shown, in both the AGS and HGC cell lines overexpressing ENO3, the erosion rate in the oe-ENO3 group was significantly higher than that in the oe-con group. This indicates that overexpression of the ENO3 gene can significantly enhance the invasive ability of gastric cancer cells.

[0156] This demonstrates that overexpression of the ENO3 gene can enhance the proliferation, migration, and invasion of gastric cancer cells.

[0157] Experiment Example 4: In MKN45 cell lines stably transfected with ENO3 knockdown, lactate levels in each group of cells were measured using a lactate assay kit, and ATP levels in each group of cells were measured using an ATP assay kit. Both the lactate and ATP assay kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0158] The results are as follows Figure 24 and Figure 25 It was found that the lactate and ATP levels in the sh-con group were significantly higher than those in the sh1-ENO3 and sh2-ENO3 groups. This indicates that knocking down the ENO3 gene can significantly reduce the lactate and ATP levels in gastric cancer cells.

[0159] Using lentivirus-mediated gene overexpression, an ENO3-overexpressing cell line was constructed in the gastric cancer cell line MKN45 and divided into three groups: a blank control group (oe-con) and an overexpression group (oe-ENO3). The blank control group (oe-con) consisted of cell lines that did not overexpress ENO3, while the overexpression group (oe-ENO3) consisted of cell lines that overexpressed ENO3. Lactate and ATP levels in the cells of each group were measured using a lactate assay kit and an ATP assay kit, respectively. Both the lactate and ATP assay kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0160] The results are as follows Figure 26 and Figure 27 It was found that the lactate and ATP levels in the oe-con group were significantly lower than those in the oe-ENO3 group. This indicates that overexpression of the ENO3 gene can significantly increase the lactate and ATP levels in gastric cancer cells.

[0161] This shows that ENO3 positively regulates glycolysis in gastric cancer cells.

[0162] Experimental Example 5: In MKN45 cell lines stably transfected with ENO3 knockdown, the expression of multiple target genes was detected using qPCR, including those from the Notch signaling pathway. HES1 , HEY1 , HEY2 Genes; NF-κB signaling pathway TNF -α、 IL - 6 , IL - 8 Genes; mTOR signaling pathway mTOR , CLN3 , HIF - 1α Genes; TGFβ signaling pathway TGFβ-Ⅰ , TGFβ-II Genes; Hedgehog signaling pathway PTCH1 , GLI1 Genes; FGF1 gene; Hippo-YAP signaling pathway CTGF , ANKRD1 and CYR61 Genes; WNT / β-catenin signaling pathway CCND1 , c-Myc , CD44 , MMP-7 Gene.

[0163] The results are as follows Figure 28 As shown, in gastric cancer cells with knocked-down ENO3, the activity of multiple signaling pathways involved in tumorigenesis and development is altered, with the most significant decrease in the activity of the WNT / β-catenin signaling pathway, whose target genes... CCND1 , c-Myc , CD44 , MMP-7 The mRNA expression level was significantly reduced.

[0164] In the MKN45 cell line overexpressing ENO3, the protein expression and phosphorylation levels of β-catenin and GSK-3β were detected by Western blot (WB), with β-actin as the reference gene.

[0165] The results are as follows Figure 29 As shown, in the MKN45 cell line overexpressing ENO3, the phosphorylation level of β-catenin at the Ser675 site was decreased, and the expression level of β-catenin was increased; the phosphorylation level of GSK-3β was significantly increased.

[0166] In the SGC-7901 cell line stably transfected with ENO3 knockdown, the expression of MMP2, MMP13, and MMP21 genes was detected by qPCR; MMP2 expression was detected by Western blot (WB). Using lentivirus-mediated gene overexpression, an SGC-7901 cell line overexpressing ENO3 was constructed; in the ENO3-overexpressing SGC-7901 cell line, the expression of MMP13 and MMP21 genes was detected by qPCR. In the MKN45 cell line stably transfected with ENO3 knockdown, MMP2 expression was detected by Western blot (WB). In the MKN45 cell line overexpressing ENO3, MMP2 expression was detected by Western blot (WB). In HGC cell lines overexpressing ENO3, the expression of MMP7, MMP13, and MMP21 genes was detected by qPCR.

[0167] The results are as follows Figures 30 to 35 As shown, ENO3 knockdown or overexpression can regulate the expression of multiple MMP molecules. MMP2, MMP7, MMP13 and MMP21 may be involved in the role of ENO3 in promoting the migration and invasion of gastric cancer cells.

[0168] Therefore, ENO3 may promote the development and progression of gastric cancer by activating the WNT / β-catenin pathway and upregulating the expression of MMPs.

[0169] Experimental Example 6: The capture efficiency of the modified immunomagnetic beads prepared in Examples 2-7 and Comparative Examples 2-6 on the gastric cancer cell line MKN45 was detected, and the specific steps are as follows: 100 fluorescently labeled gastric cancer cell line MKN45 cells were mixed with 1×10 6Individual peripheral blood mononuclear cells were mixed, 1 mL of phosphate buffer was added, and 250 μL of modified immunomagnetic beads were added and mixed for 30 min. The mixture was then placed on a magnetic rack for 5 min, the suspension was discarded, and the cells were washed three times with washing buffer. The cells were removed from the rack, and 200 μL of buffer was added again and mixed. The mixture was centrifuged at 500 rpm for 5 min, the pellet was retained, and 1 mL of cell dissociation buffer was added and incubated for 5 min. The cells were then placed on a magnetic rack for 5 min, and the supernatant was collected to obtain tumor cells. The cells were observed under a microscope, photographed, and their number was quantified. The capture efficiency was calculated using the following formula: Capture efficiency (%) = (Total number of gastric cancer cells - Number of gastric cancer cells in supernatant) / Total number of gastric cancer cells × 100%.

[0170] Table 1. Capture efficiency (%)

[0171] The results are shown in Table 1. Compared with Comparative Examples 2-3, the capture efficiency of Examples 2-7 and Comparative Examples 4-6 was significantly increased. This is because the modified immunomagnetic beads were different. Comparative Example 2 used commercially available carboxyl magnetic beads coupled with ENO3 antibody to obtain modified immunomagnetic beads, and Comparative Example 3 used commercially available amino magnetic beads coupled with ENO3 antibody to obtain modified immunomagnetic beads. However, Examples 2-7 and Comparative Examples 4-6 used the modified magnetic beads prepared according to this invention coupled with ENO3 antibody to obtain modified immunomagnetic beads. This indicates that using the modified magnetic beads prepared according to this invention coupled with ENO3 antibody to obtain modified immunomagnetic beads, and using them to enrich trace amounts of tumor cells in peripheral blood samples from gastric cancer patients, can effectively improve the capture efficiency of tumor cells.

[0172] Compared with Comparative Example 4, the capture efficiency of Example 2 was significantly increased. This is because the modified magnetic beads used in the preparation of the modified immunomagnetic beads were different. Comparative Example 4 used a polymeric substance with methacrylic acid as the structural unit to coat oleic acid-modified Fe3O4 composite nanoparticles to obtain modified magnetic beads, while Example 2 used a polymeric substance with methacrylic acid and 2-[[(butylamino)carbonyl]oxo]acrylate as the structural units to coat oleic acid-modified Fe3O4 composite nanoparticles to obtain modified magnetic beads. This indicates that using the modified immunomagnetic beads prepared in Example 2 of this invention, and applying them to enrich trace amounts of tumor cells in peripheral blood samples from gastric cancer patients, can effectively improve the capture efficiency of tumor cells.

[0173] Compared with Example 2 and Comparative Example 5, the capture efficiency of Examples 3-6 was increased, with Example 3 showing the best capture effect. This indicates that using polymers with methyl methacrylate, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate, and N-allyl-P-methoxyaniline as structural units to coat oleic acid-modified Fe3O4 composite nanoparticles to prepare modified magnetic beads, which are then coupled with ENO3 antibody to form modified immunomagnetic beads for the enrichment of tumor cells, can effectively improve the capture efficiency for gastric cancer patients. Furthermore, when the mass ratio of methyl methacrylate to N-allyl-P-methoxyaniline is in the range of 1:0.1-0.25, the capture efficiency for tumor cells is better; too high or too low a proportion of N-allyl-P-methoxyaniline will lead to poor capture efficiency.

[0174] Compared with Example 3 and Comparative Example 6, the capture efficiency of Example 7 was significantly increased. This indicates that by coating oleic acid-modified Fe3O4 composite nanoparticles with polymers consisting of methacrylic acid, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate, N-allyl-P-methoxyaniline, and methyl 3-allyl-2-hydroxybenzoate as structural units, modified magnetic beads were prepared, and then coupled with ENO3 antibody to form modified immunomagnetic beads for the enrichment of tumor cells, which can further improve the capture efficiency of gastric cancer patients.

[0175] Experiment Example 7: The expression of ENO3 in peripheral blood samples from 54 patients with early-stage gastric cancer at Zhejiang Cancer Hospital was detected using the detection methods for ENO3, a gastric cancer biomarker, as described in Examples 2-7 and Comparative Examples 1-6, respectively. The presence of a peak in qRT-PCR amplification was used as the positive criterion, and the absence of a peak was used as the negative criterion. The positive detection rate was calculated using the following formula: Positive detection rate (%) = (Number of positive results / Total number of positive results) × 100%.

[0176] Table 2 Positive Detection Rate (%)

[0177] The results are shown in Table 2. Compared with Comparative Example 1, the positive detection rates of Examples 2-7 and Comparative Examples 2-6 were significantly higher. This is because, in the detection of the gastric cancer marker ENO3, Comparative Example 1 did not perform a tumor cell enrichment step and directly performed qRT-PCR detection on whole blood samples, while Examples 2-7 and Comparative Examples 2-6 introduced a tumor cell enrichment step before qRT-PCR detection. This indicates that using modified immunomagnetic beads to enrich trace amounts of tumor cells in peripheral blood samples from gastric cancer patients, followed by qRT-PCR detection of the gastric cancer marker ENO3 expression, can effectively improve the positive detection rate of gastric cancer patients and reduce false negative results caused by excessively low target signal.

[0178] Compared with Comparative Examples 2-3, the positive detection rates of Examples 2-7 and Comparative Examples 4-6 were significantly increased. This is due to the different modified immunomagnetic beads used. Comparative Example 2 used commercially available carboxyl magnetic beads coupled with ENO3 antibody to obtain modified immunomagnetic beads, and Comparative Example 3 used commercially available amino magnetic beads coupled with ENO3 antibody to obtain modified immunomagnetic beads. However, Examples 2-7 and Comparative Examples 4-6 used the modified magnetic beads prepared according to this invention coupled with ENO3 antibody to obtain modified immunomagnetic beads. This indicates that, under the premise of using cell enrichment steps, the modified immunomagnetic beads prepared according to this invention have a significantly higher positive detection rate for ENO3 in clinical samples compared to using commercially available magnetic beads. This may be because the modified immunomagnetic bead structure prepared according to this invention has a more efficient enrichment ability for trace amounts of tumor cells in peripheral blood.

[0179] Compared with Comparative Example 4, the positive detection rate of Example 2 was significantly higher. This is because the modified magnetic beads used in the preparation of the modified immunomagnetic beads were different. Comparative Example 4 used a polymeric substance with methacrylic acid as the structural unit to coat oleic acid-modified Fe3O4 composite nanoparticles to obtain modified magnetic beads, while Example 2 used a polymeric substance with methacrylic acid and 2-[[(butylamino)carbonyl]oxo]acrylate as the structural units to coat oleic acid-modified Fe3O4 composite nanoparticles to obtain modified magnetic beads. This indicates that using the modified immunomagnetic beads prepared in Example 2 of this invention, and using them to enrich trace amounts of tumor cells in peripheral blood samples from gastric cancer patients, can effectively improve the positive detection rate of gastric cancer patients.

[0180] Compared with Example 2 and Comparative Example 5, the positive detection rates of Examples 3-6 were all increased, with Example 3 showing the best positive detection effect. This indicates that using polymers with methyl methacrylate, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate, and N-allyl-P-methoxyaniline as structural units to coat oleic acid-modified Fe3O4 composite nanoparticles to prepare modified magnetic beads, which are then coupled with ENO3 antibody to form modified immunomagnetic beads for the enrichment of tumor cells, can effectively improve the positive detection rate of gastric cancer patients. Furthermore, the detection effect is better when the mass ratio of methyl methacrylate to N-allyl-P-methoxyaniline is in the range of 1:0.1-0.25; both excessively high and low proportions of N-allyl-P-methoxyaniline will result in poor positive detection rates.

[0181] Compared with Example 3 and Comparative Example 6, the positive detection rate of Example 7 was significantly increased. This indicates that modified magnetic beads were prepared by coating oleic acid-modified Fe3O4 composite nanoparticles with polymers consisting of methacrylic acid, ethyl 2-[[(butylamino)carbonyl]oxo]acrylate, N-allyl-P-methoxyaniline, and methyl 3-allyl-2-hydroxybenzoate as structural units. These modified magnetic beads were then coupled with ENO3 antibody to form modified immunomagnetic beads, which were used for the enrichment of tumor cells, thereby further improving the positive detection rate in subsequent ENO3 detection.

[0182] Experimental Example 8: The morphology of the modified immunomagnetic beads prepared in Example 7 was observed using a scanning electron microscope.

[0183] The results are as follows Figure 36 As shown, the modified immunomagnetic beads prepared in this invention were successfully synthesized. The modified immunomagnetic beads have a spherical structure, with multiple Fe3O4 particles partially encapsulated within the microspheres. The modified immunomagnetic beads exhibit a certain degree of aggregation, which may help improve contact efficiency during cell capture.

[0184] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0185] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any changes and modifications made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A reagent for detecting gastric cancer markers, characterized in that, include: Upstream and downstream primers for a gastric cancer marker; the gastric cancer marker is enolase 3; the upstream primer sequence is 5'-GAACTCCGAGATGGAGACAAAG-3', and its nucleotide sequence is shown in SEQ ID NO.1; the downstream primer sequence is 5'-GGACCTAGAGTCTTGTTGATGTG-3', and its nucleotide sequence is shown in SEQ ID NO.2; The detection reagent also includes modified immunomagnetic beads. The modified immunomagnetic beads are prepared by first mixing an alkenyl derivative, divinylbenzene, and sodium dodecyl sulfate, then adding deionized water and oleic acid-modified Fe3O4 composite nanoparticles for ultrasonic dispersion, and then reacting with acetic acid and H2O2 solution under a nitrogen atmosphere to obtain modified magnetic beads. Subsequently, enolase 3 antibody is coupled to the surface of the modified magnetic beads using EDC-NHS coupling technology to obtain modified immunomagnetic beads. The alkenyl derivative is methacrylic acid and ethyl 2-[[(butylamino)carbonyl]oxo]acrylate in a mass ratio of 1:0.5-2.

2. The detection reagent for gastric cancer markers according to claim 1, characterized in that, The alkenyl derivative further includes N-allyl-P-methoxyaniline, wherein the mass ratio of methacrylic acid to N-allyl-P-methoxyaniline is 1:0.1-0.

25.

3. The detection reagent for gastric cancer markers according to claim 1 or 2, characterized in that, The alkenyl derivative further includes methyl 3-allyl-2-hydroxybenzoate, wherein the mass ratio of methacrylic acid and methyl 3-allyl-2-hydroxybenzoate is 1:0.3-1.

4. The detection reagent for gastric cancer markers according to claim 1, characterized in that, The ratio of the alkenyl derivative to divinylbenzene is 1g:0.01-0.1mL.

5. The detection reagent for gastric cancer markers according to claim 1, characterized in that, The ratio of divinylbenzene to sodium dodecyl sulfate is 1 mL: 0.2-0.5 g.

6. The detection reagent for gastric cancer markers according to claim 1, characterized in that, The mass ratio of sodium dodecyl sulfate and oleic acid-modified Fe3O4 composite nanoparticles is 1:0.5-2.

7. The detection reagent for gastric cancer markers according to claim 1, characterized in that, The volume ratio of divinylbenzene to acetic acid is 1:0.1-0.

5.

8. The detection reagent for gastric cancer markers according to claim 1, characterized in that, The mass concentration of the H2O2 solution is 20-40%, and the volume ratio of divinylbenzene to H2O2 solution is 1:0.1-0.5.

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